Method for selecting mevalonate synthesis modulators using cells derived from human pluripotent cells

ABSTRACT

The invention provides a method for selecting pharmaceutical compounds that enhance inhibition of mevalonate synthesis in which mesodermal stem cells (MSC type) that are derived from human pluripotent cells or from induced stem cell are contacted with pharmaceutical compounds to be tested in the presence of an inhibitor of mevalonate synthesis. Pharmaceutical compounds are then identified which enhance cell toxicity in the presence of the inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/805,547, filed Feb. 5, 2013, which is now U.S. Pat. No. 8,759,022,which is a national stage application filed under 35 USC 371 ofInternational Application No. PCT/IB2011/052680, filed Jun. 20, 2011,which claims priority from EP patent application 10166631.1 filed Jun.21, 2010, the entire contents of which are incorporated herein byreference.

FIELD OF THE INVENTION

The synthesis of mevalonate is involved in many biological processessuch as the synthesis of cholesterol, as well as the prenylation ofproteins. The different steps of the metabolic route have beenidentified. The limiting HMG CoA reductase, an enzyme for the synthesisof mevalonate, is a major therapeutic target. Statins which arecompetitive inhibitors of HMG CoA reductase form an important class oftherapeutic molecules. The first clinical application of these moleculesis the reduction of lipoproteins of the LDL types in the general bloodstream. Moreover, these molecules have pleiotropic effects, some ofwhich are independent of the control of the synthesis of cholesterol andprobably related to secondary modifications of the proteins involved incell signaling. Statins improve the function of endothelial cells,stabilize atheroma plates, have anti-inflammatory functions, play a rolein bone resorption, reduce the risks of dementia, control cellproliferation of certain normal and tumoral cells.

Moreover, statins have significant toxicity for certain tissues such asmuscle tissue, which limits the clinical use thereof.

Cell tests allowing high throughput screening of compounds interferingwith the metabolism of mevalonate should allow:

-   -   Identification of novel functional inhibitors of the synthesis        of mevalonate.    -   Providing drug interactions having the consequences of        increasing cell toxicity, in particular muscular toxicity.    -   Identification of molecules preventing tissue toxicity, in        particular muscular toxicity.    -   Identification of molecules preventing muscle atrophy.    -   Identification of molecules selectively controlling the        proliferation of normal and tumoral cells, including tumoral        stem cells.

The present invention relates to methods for screening moleculesinvolved in the synthesis of mevalonate notably by using differentiatednormal MSC cells or of the MSC type, derived from pluripotent cells.

SUMMARY OF THE INVENTION

The efficiency of high throughput screening by using cell tests, isdependent on the specificity, on the sensitivity, on the robustness andavailability of the cell tests used. This issue is particularlyimportant for normal human cells which are the most suitable models forscreening therapeutic molecules for the human species. Indeed, theavailability of normal human cells is in particular limited for certaincell types such as cardiac and neural cells. On the other hand, normalhuman cells have limited replication capacity restricting their use forhigh throughput screening. Presently, the large majority of the cellsused for high throughput screenings of the HTS type are either non-humancells or else transformed or tumoral human cells which decreases therelevance of the isolated molecules in this type of screening.

The present invention relates to methods for high throughput screeningof molecules involved in the synthesis of mevalonate by usingdifferentiated normal MSC cells or of the MSC type derived frompluripotent cells. The invention also relates to methods for producingdifferentiated normal cells derived from pluripotent cells. Theinvention also relates to methods for high throughput screening usingdifferentiated normal cells derived from pluripotent cells bycontrolling the synthesis of mevalonate by the presence ofpharmacological inhibitors.

The object of the present invention is notably:

-   -   1. A method for producing cells of the MSC type from human        pluripotent cells comprising a step for cultivating human        pluripotent cells in a culture medium comprising:        -   a. one or several growth factors selected from FGFs, HGFs,            PDGFs, EGF, herugulins and VEGFs        -   b. one or more antioxidants selected from ascorbic acid and            its derivatives, vitamin E and N-acetylcysteine    -   2. A method for producing cells of the MSC type from induced        pluripotent stem cells comprising a step for cultivating the        induced stem cells in a culture medium comprising:        -   a. one or more growth factors selected from FGFs, HGF,            PDGFs, EGF, herugulins and VEGFs        -   b. one or more antioxidants selected from ascorbic acid and            its derivatives, vitamin E and N-acetylcysteine.    -   3. The method according to object 1 or 2 characterized in that        the culture medium comprises:        -   a. FGF; and        -   b. ascorbic acid or one of its derivatives.    -   4. The method according to object 3 characterized in that the        culture medium comprises:        -   a. FGF2; and        -   b. ascorbic acid and/or ascorbic acid 2-phosphate.    -   5. The method according to object 4 characterized in that the        culture medium comprises FGF2 at a final concentration of 10        ng/mL and ascorbic acid 2-phosphate at a final concentration of        1 mM.    -   6. The method according to the preceding objects, characterized        in that the produced cells of the MSC type express one or more        of the CD73, CD29, CD44, CD166 or CD 105 markers.    -   7. The method according to the preceding objects characterized        in that the produced cells of the MSC type express SSEA4.    -   8. A method for selecting pharmaceutical compounds affecting        mevalonate or cholesterol, comprising a step for putting the        cells obtained by the methods of objects 1 to 7 into contact        with the pharmaceutical compounds to be tested.    -   9. A method for selecting pharmaceutical compounds modulating        the metabolic routes of mevalonate or cholesterol, comprising a        step for putting the cells obtained by the methods of objects 1        to 7 into contact with the pharmaceutical compounds to be        tested.    -   10. A method for selecting pharmaceutical compounds enhancing        the effects of inhibition of the synthesis of mevalonate,        comprising a step for putting the cells obtained by the methods        of objects 1 to 7 into contact with the pharmaceutical compounds        to be tested.    -   11. A method for selecting pharmaceutical compounds compensating        for the effects of the inhibition of the synthesis of        mevalonate, comprising a step for putting the cells obtained by        the methods of objects 1 to 7 into contact with the        pharmaceutical compounds to be tested.    -   12. A method for selecting pharmaceutical compounds, functional        inhibitors of the synthesis of mevalonate and of cholesterol,        comprising a step for putting the cells obtained by the methods        of objects 1 to 7 into contact with the pharmaceutical compounds        to be tested.    -   13. A method for selecting pharmaceutical compounds having a        toxic effect on muscular tissue, comprising a step for putting        the cells obtained by the methods of objects 1 to 7 into contact        with the pharmaceutical compounds to be tested.    -   14. A method for selecting pharmaceutical compounds protecting        against muscle atrophy or sarcopenia, comprising a step for        putting the cells obtained by the methods of objects 1 to 7 into        contact with the pharmaceutical compounds to be tested.    -   15. A method for selecting antitumoral pharmaceutical compounds        comprising a step for putting the cells obtained by the methods        of objects 1 to 7 into contact with the pharmaceutical compounds        to be tested.    -   16. Cells obtained by the methods of objects 1 to 7 for        repairing bone tissue.    -   17. Cells obtained by the methods of objects 1 to 7 for        repairing cartilage.    -   18. The use of cells obtained by the methods of objects 1 to 7        for obtaining white or brown adipocytes.    -   19. The use of cells obtained by the methods of objects 1 to 7        for obtaining immunomodulating cells.

DESCRIPTION OF THE INVENTION

The present invention relates to methods for high input screening ofmolecules involved in the synthesis of mevalonate by usingdifferentiated normal MSC cells or of the MSC type derived frompluripotent cells. One of the goals of this novel method is to increaserobustness, specificity and sensitivity of the high throughputscreenings by associating sources of normal human cells available in anunlimited amount and modulators of the synthesis of mevalonate. Thespecificity, sensitivity, robustness and availability of cell systemsdetermine the relevance of high throughput screenings.

Very frequently, the cells used for high throughput screenings are cellsof rodents (rat, mouse) or transformed human cells. Unlike many rodentcells which have significant probabilities of spontaneoustransformation, normal human cells have finite growth capacities andenter senescence after about 50 cell divisions. The extended growth ofhuman cells is accompanied by phenotype instability. These propertiesconsiderably limit the use of normal human cells for high throughputscreenings.

The existence of human pluripotent stem cells gives the possibility ofhaving access to normal human cells without any limitations. There existtwo types of pluripotent cells: embryonic stem cells (hES) and inducedstem cells (iPS). The essential characteristics of pluripotent cells arethe capability of self-renewal without any limitation and thepossibility of differentiation into all the types of cells making up anentire organism.

The possibility since the work of J. Thompson (Thompson et al. 1998) ofusing human embryonic stem cells (hES) has opened a large number ofpossibilities. Experimentally, human embryonic stem cells have alloweddevelopment of differentiation procedures and isolation of many types ofcells (cardiac cells, smooth muscle cells, neural cells, keratinocytes,hematopoietic cells, insulin-producing cells). However, the isolation ofembryonic stem cells is a technology which requires significant know-howand which is dependent on accessing sensitive and limited biologicalmaterial: supernumerary embryos. Further, embryonic stem cells raisemany ethical issues which are solved differently depending on thenational states. Ethical, regulatory and industrial property issuespresently limit the use of embryonic stem cells.

The discovery of iPS cells by the team led by S. Yamanaka (Takahashi K.& Yamanaka S. 2006) has shown the reversible nature of the latter andthe reduced number of genes for which <<forced>> co-expression may causededifferentiation and reprogramming of cells of very diverse types intoiPS cells having properties very similar to ES cells: self-renewal,differentiation capability into all cell types making up an organism andcapability of forming teratomas once ejected into tolerant animals.There does not seem to exist any limitations for obtaining iPS. Indeed,primary cells, immortalized cells, skin or lung fibroblasts,hepatocytes, pancreas cells, lymphocytes and neural precursors were ableto be <<reprogrammed>> into iPS cells. iPS cells stemming from cells ofpatients affected with many pathologies such as SMA, ALS, Parkinson's orDuchenne's Dystrophy have also been produced (Park et al. Et 2008). Thisapproach therefore allows isolation of iPS cells from easily accessiblebiological materials. Finally, it must be added that these very numerousstudies clearly show the qualitative robustness of this technology.

The first part of the invention relates to the production ofdifferentiated MSC cells or of the MSC type derived from pluripotentcells sensitive to the inhibitors of HMG CoA reductase, a key enzyme inthe synthesis of mevalonate. Former studies have shown that cellsderived from muscular tissue, muscle precursor cells (MPC), haveparticular sensitivity to statins. Indeed, the presence of statinspecifically inhibits the growth of this type of cells. The describedmethod of the invention allows production of differentiated cellssensitive to inhibitors of HMG CoA reductase derived from pluripotentcells: hES cells and iPS cells. The production method is a two-stepmethod, the first step being a differentiation method from pluripotentcells and the second being a method for amplifying and preservingthereby differentiated cells.

The different steps of the production method are described below. Theprinciple of this method is to initiate differentiation by mechanical orenzymatic disassociation of non-differentiated cell clusters stemmingfrom pluripotent cells and by sowing on a cell support consisting ofcollagen or of its derivatives such as gelatin. Once the cells aredifferentiated, the cells are amplified in a medium comprising growthfactors from the FGF family and antioxidants such as the derivatives ofascorbic acid. For this purpose, other growth factors may be used suchas HGF (Hepatocyte Growth Factor), various PDGFs (Platelet DerivedGrowth Factor), factors from the EGF (Epidermal Growth Factor) family,from the VGEF family, herugulins. With this method, it is possible toproduce differentiated cells without any limitations on amounts, whichhave a stable phenotype. These cells have a normal phenotype which ischaracterized by a normal karyotype and by finite growth.

Definitions of the Cell Types

Pluripotent Stem Cells

Pluripotent cells are characterized by two properties: the self-renewalcapability and that of forming all the cell types making up an adultorganism. These cells by differentiation may give cells of the freeembryonic layers (ectodermis, endodermis, mesodermis). There essentiallyexist two types of pluripotent cells, embryonic stem cells and cellsinduced to pluripotency.

Embryonic Stem Cells (ES Cells).

Embryonic stem cells are pluripotent stem cells stemming from an embryoin the blastocyst stage. Isolation of these cells required resorting toembryos. Today, there exist worldwide very numerous lines of embryonicstem cells which are available.

Induced Pluripotent Stem Cells (iPS Cells)

Induced pluripotent stem cells (iPS) stem from reprogramming adultsomatic cells into pluripotent cells, are, for the moment in the largemajority of the cases, obtained by transfer of genes required forreprogramming. Among these genes, genes expressed by ES cells are foundsuch as Sox2 and Oct4 and genes controlling proliferation such as cMyc,Lin 28 and Klf4. iPS cells have the essential properties of ES cells. Inthe near future, it will be possible to obtain iPSes by using otherapproaches such as transfer of proteins, treatment with small molecules.

Mesodermal Progenitor Cells (MePC)

<<MSC>> cells are derived from pluripotent cells (ES and iPS). Thesecells belong to the mesodermal layer, are dependent for their growth onattachment to a substrate and have a significant but finite growthcapacity. <<MSC>> cells express a series of membrane markers such asCD44, CD29, CD73, CD105 and SSEA4 and are under certain experimentalconditions able to form bone tissue, cartilage tissue and adiposetissue.

Mesodermal Stromal or Stem Cells (MSC Cells).

In adults, MSCs may be isolated from very numerous tissues. The firstMSC cells were isolated from bone marrow. In a second phase, MSC cellswere found in almost all the investigated tissues. These cells belong tothe mesodermal layer and are dependent for their growth on attachment toa substrate and have a finite growth capacity. MSC cells express aseries of membrane markers such as CD44, CD29, CD73 and under certainexperimental conditions are capable of forming bone tissue, cartilagetissue and adipose tissue.

MSC or MSC Type Cells

MSC or MSC type cells mean mesodermal stem cells also known under thename of multipotent stroma cells, as well as cells having the samebiological characteristics. MSC or MSC type cells in a non-limiting wayinclude mesodermal progenitor cells (MePC) and mesodermal stromal orstem cells of adult tissue (MSC).

Muscle Progenitor Cells (<<MPC>>).

MPC cells stem and are isolated from muscle tissue. These cells areresponsible for maintaining the muscle function and control the repairof muscle tissue. MPCs are capable of self-renewal in a limiting way andof forming muscle tissue ex vivo and in vivo.

Description of the Procedures for Isolating and Producing Cells forScreening, Derived from Human Pluripotent Cells (hES and iPS).

The cells may be produced from non-differentiated hES cells or iPScells. The production conditions are the following and are identical forboth cell types. The islets of non-differentiated cells which may beidentified by their morphological characteristics form the basis of thecell material for producing cells.

Non-differentiated cell islets which may be identified on morphologicalcriteria or on expression criteria such as the expression of Tra1-60 orthe expression of GFP under the control of a gene promoter associatedwith maintaining pluripotency such as Oct4/pouf5 are mechanicallydissociated under control of a binocular magnifier, by means of aneedle. Mild enzymatic dissociation by using enzymes of the collagenaseand trypsin type may also be used. It is also possible to add chelatingagents of divalent ions such as EDTA and EGTA. In the latter case, theuse of an inhibitor of the Rhoa protein (Rock inhibitor) allows betterrestarting of the thereby dissociated cells. The small clusters of cellsobtained after dissociation are automatically counted or by means of anhematocytometer and the viabilities are estimated by using trypan blueexclusion or propidium iodide marking techniques. The following step isformed by the sowing of the cell aggregates on cell supports coveredwith collagen or with its derivatives such as gelatin. Otherconstituents of the extra-cellular matrix such as fibronectin, laminin,vitronectin, or synthetic compounds such as poly-ornithine may be usedfor this step. The cells are then cultivated in the presence of aculture medium of the DMEM type supplemented with glutamine, with amixture of non-essential amino acids, of beta-mercaptoethanol and ofbovine serum or from another origin such as human serum. The operationmay be carried out in a completely synthetic medium without any proteinof animal origin.

During this invention, it was able to be shown that the association ofFGF with antioxidants such as derivatives of ascorbic acid increasedboth the robustness of the production method, the production kineticsand the amount of produced cells. By using this approach combining FGFwith ascorbic acid 2-phosphate, differentiated cells derived frompluripotent cells were able to be produced in all the experiments withthe different types of pluripotent cells whether these be hES cells oriPSes. Five days after sowing, it is possible to distinguish individualcells adhering to the cell substrate which emerge from cell clusters andthen enter cell divisions. This is the first step of morphologicaldifferentiation. Between day 10 and 20, the cells are transplanted byusing enzymatic solutions of the trypsin type combined with chelatingagents of the EDTA types. It is also possible to use mechanicaldissociation. The thereby obtained cells are sown on cell supportscovered with collagen or with its derivatives of the gelatin type. Likefor the first step, it is possible to use another type of substrate.This second step is the cell amplification step which allows an increasein the number of cells and homogenization of the thereby obtained cellpopulation. As soon as the first transplantation, the presence of FGFassociated with ascorbic acid 2-phosphate allows an increase in thenumber of cells by a factor 2 and then during the passages, the numberof cells considerably increases in the presence of this combination. Thedifference may then attain several log factors. It is also possible toproduce this type of cells in synthetic media containing FGF andantioxidants such as ascorbic acid and its derivatives. It is remarkablethat the presence of FGF without any antioxidant does not allow anincrease in the number of cells derived from pluripotent cells. Thethereby obtained cells are preserved by standard methods such ascryopreservation with DMSO as cryopreservation agents. For this step, itis also possible to use glycerol, trehalose, glycine and arbutin ascryoprotective agents as well as commercial solutions. The preservationmay be effected by vitrification. Cryopreservation of the produced cellshas excellent yield and does not modify the biological parameters suchas cell growth or the expression of surface markers. Under theseproduction conditions, the cells keep their normal nature while havingfinite growth and a normal karyotype.

The described differentiation and amplification methods for the therebyproduced cells have a similar efficiency for cells derived from hES oriPS. In both cases, the presence of the combination of FGF associatedwith ascorbic acid 2-phosphate in the culture medium dramaticallyincreases the efficiency of the cell differentiation and amplificationmethods.

Characterization of Differentiated Cells Derived from Human PluripotentCells (hES and iPS) for Screening.

The first characterization steps are based on functional criteria suchas the growth capacity, adhesion to the substrates and on morphologicalcriteria. Morphologically, the produced cells have characteristicssimilar to those of mesenchymal cells. Their growth is dependent on theattachment to a substrate. Molecular analyses carried out show that theexpression of the genes associated with the characteristic pluripotencycondition of pluripotent cells such as Oct4, Nanog are undetectable incells produced by RT-PCR techniques.

Analysis of surface markers by flow cytometry of the produced cellsshows an absence of expression of SSEA3, of Tra-1-60, of Tra-1-80 whichare membrane determinants associated with the pluripotency condition anda presence of membrane determinants such as CD73, CD44, CD166, CD105 andCD29. The expression of these markers is also present on multipotentstroma cells (MSC). MSC cells were individualized about ten years ago.The cells produced by the method of the invention therefore sharecertain properties of MSCs:

-   -   Adhesion to the substrates    -   High but finite growth potential    -   Preservation by freezing    -   Expression homogeneity for the CD73, CD29, CD44, CD166 markers.    -   Osteogenic differentiation potential.        The Combined Presence of FGF2 and of Ascorbic Acid 2-Phosphate        in the Culture Medium Increases the Growth Capacity and the        Expression of SSEA4 of the Cells Produced by the Method of the        Invention.

In the method of the invention, with the presence of FGF2 and ofascorbic acid 2-phosphate, it is possible to produce differentiatedcells from pluripotent cells. This increase in the growth capacities isassociated with the specific expression of a membrane marker SSEA4. Thismembrane marker is also expressed by pluripotent cells and certain(neural or mesodermal) progenitor cells. It is remarkable to note thatFGF2 and ascorbic acid 2-phosphate individually do not give thepossibility of inducing expression of SSEA4 or of significantlyincreasing the proliferation capabilities. On the other hand, thepresence of FGF2 and of ascorbic acid 2-phosphate together considerablyincreases the growth capacities and the expression of SSEA4 withoutmodifying the expression of other markers associated with totipotencylike Tra-1-60, Tra-1 80, SSEA3 and SSEA1. With the method of theinvention, it is possible to homogeneously produce differentiated cellswhich express SSEA4. Under these production conditions described by themethod of the invention, the differentiated cells have a significantproliferation capacity which is associated with the expression of SSEA4.

The Cells Produced by the Method of the Invention have a SensitivitySpecific to the Inhibitors of the Synthesis of Mevalonate Like theInhibitors of HMG CoA Reductase.

Mevalonate is a precursor indispensable for the synthesis of metabolitecholesterol, key of cell activity. Statins, steric inhibitors of HMG CoAreductase (an enzyme which controls the synthesis of mevalonate) blockthe production of mevalonate and thus the production of cholesterol.With provision of mevalonate by the extra-cellular medium, it ispossible to restore the production of cholesterol in spite of thepresence of statin.

These properties give the possibility of building cell tests notably forpredicting and analyzing the toxicity of the inhibitors of the synthesisof mevalonate. Certain cell types like MPC cells are particularlysensitive to statins. In this cell type, the statins are toxic and thistoxicity is abolished by the presence of mevalonate in the presence ofextra-cellular medium. It is thus possible to show by a simple cell testthat statins are toxic through a mechanism which involves the synthesisof mevalonate.

Recent examples with the inhibitors of COX 2 and of HMG CoA reductaseshow that underestimating toxicological issues may have considerableconsequences from a human, health and economical point of view. Definingpredictive toxicology systems is therefore a critical goal. Theinhibitors of HMG CoA reductase, including statins, have particulartoxicity for muscle tissue. This toxicity may range from simple aches torhabdomyolyses which may cause death.

Human muscle precursor cells (MPCs) are good indicators of muscletoxicity. Indeed, cell growth of MPCs is inhibited by the presence ofstatin in the growth medium. This inhibition is dose-dependent. Thepresence of mevalonate gives the possibility of totally lifting theinhibition of growth. This inhibition is therefore accomplished throughthe inhibition of HMG CoA reductase, an enzyme responsible for theproduction of mevalonate, a precursor in the synthesis of cholesterol.

The cell produced from pluripotent cells according to the method of theinvention have a sensitivity comparable to the MPCs for the inhibitorsof HMG CoA reductase. Indeed, the presence of inhibitors of HMG CoAreductase like novastatin or simvastatin in the culture medium reducesthe number of differentiated cells derived from pluripotent cells in adose-dependent way. This inhibition is raised by the presence ofmevalonate. The thereby observed inhibition of growth is therefore dueto the inhibition of the enzyme responsible for the production ofmevalonate. The cells produced from embryonic stem cells and thoseproduced from induced pluripotent cells have the same type ofsensitivity to the inhibitors of HMG CoA reductase and to mevalonate.

The technology of iPSes gives the possibility of producing pluripotentcells from any individuals. The method of the invention gives thepossibility of generating differentiated cells derived from iPS cellsfrom patients having exacerbated sensitivity to the inhibitors of thesynthesis of mevalonate. These cell tools will be useful forunderstanding the toxicity mechanisms and isolating markers forpreventing and tracking this toxicity.

The cells produced by the method of the invention allow high throughputscreening of the synthesis of mevalonate.

High throughput screening of compounds requires the possibility ofcultivating the cells in formats compatible with this technology such as96- or 384-well multiwell plates. On the other hand, the use of rapidsystems for reading out the results, compatible with analysis robots, isalso required. The cells produced by the method of the invention may becultivated in 96- and 384-well multiwell plates. Under these conditions,the variation coefficients of cell counts are small. In a first phase,the number of cells obtained in multiwells was determined by directlycounting the cells after marking by using image analysis systems. Theseanalysis systems are accurate, but relatively slow. Tests based on theamount of ATP and mitochondrial activity were therefore developed. Itwas possible to demonstrate that for the cells produced by the method ofthe invention, there exists a linear relationship between the measuredamount of ATP or mitochondrial activity and the number of cells perwell. In other words, the amount of ATP or the measured mitochondrialactivity is a measurement of the number of cells. A bank of chemicalmolecules of more than 1,200 compounds representing the main activeproducts used in human clinical practice was screened.

The cells described by the method of the invention gave the possibilityof screening this bank. These cells were cultivated in 96- and 384-wellmultiwells for three days in the presence and in the absence ofmevalonate and in the presence of the different compounds of the bank.All these experiments were carried out with robots allowing distributionof the compounds and cultivation of the cells in a sterile medium. Thecompounds were selected on the following criteria: decrease the numberof cells in the absence of mevalonate and maintaining the number ofcells in the presence of mevalonate. With these criteria it is possibleto sort out the molecules for which the toxicity is related to theinhibition of the synthesis of mevalonate. In all the screenings carriedout, the inhibitors of HMG CoA reductase, a limiting enzyme for thesynthesis of mevalonate used in human clinical practice, present in thebank, were able to be selected. These experiments were able to show thatthe cells produced according to the method of the invention allowscreening of the inhibitors of the synthesis of mevalonate in a robustway by using high throughput screening technologies. This proof offeasibility gives the possibility of contemplating screenings with thethereby described systems of banks representing several hundred thousanddifferent compounds. It should also be noted that the molecules aresorted out on a functional criterion, cell toxicity due to theinhibition of the synthesis of mevalonate. These types of tests willgive the possibility of isolating other classes of inhibitors of thesynthesis of mevalonate. With this system, it is possible to isolatenovel inhibitors on their functional capacity independently of theirdirect interaction with HMG CoA reductase.

The cells produced by the method of the invention allow high throughputscreening of compounds protecting against the toxic effect of theinhibitors of the synthesis of mevalonate. The toxic effects of theinhibitors of the synthesis of mevalonate limit the use of this type ofcompound in clinical practice. Indeed, many secondary effects onmuscular tissue are observed, most of which are limited and reversible.Nevertheless, in many cases, around 20% of the treated patients havemuscular pains (myalgias, cramps). These effects very frequently causeinterruptions in the treatment. Identifying compounds which may reducethe toxic effect of the inhibitors of the synthesis of mevalonate is agoal which may have clinical consequences. With this in mind, the screenis made by using the cells produced by the method of the invention whichare treated with inhibitors of the synthesis of mevalonate such as theinhibitors of HMG CoA reductase. The screening conditions are producedas earlier and the cells are treated with an inhibitor of HMG CoAreductase, simvastatin or all the molecules of this drug class. Thecompounds are sorted according to their capability of reducing the toxiceffects of simvastatin. By using this approach, about ten compounds wereable to be selected in a bank of more than 1,200 compounds. These arepotential candidates for reducing the toxic effects of this class oftherapeutic molecules.

On the other hand, it was shown that the inhibitors of HMG CoA reductaseinduce the expression of a protein, atrogin, a protein involved inmuscular atrophy phenomena. Muscular atrophy is associated with manypathologies (neuromuscular diseases, cancer, HIV, kidney failure, ageingor immobilization) and is per se a poor prognosis factor. The therebydescribed screen also allows isolation of compounds which may limitmuscle atrophy. Indeed, treatment with inhibitors of HMG CoA reductaseof the cells produced by the method of the invention gives thepossibility of reproducing in the culture dish, a model of controlledcell atrophy. The compounds which limit toxicity, for the cells producedby the method of the invention, in the presence of inhibitors of HMG CoAreductase, are potential candidates for reducing muscular atrophy andtreating sarcopenia. Sarcopenia is the loss of muscles due to ageing, toa neurological disease, to a viral disease (for example AIDS) or to atumoral disease. This is a frequent disease for which there does notexist yet any specific therapeutic solutions.

The cells produced by the method of the invention allow high throughputscreening of compounds enhancing the toxic effect of the inhibitors ofthe synthesis of mevalonate.

The possibility of predicting compounds enhancing cell toxicity in thepresence of inhibitors of the synthesis of mevalonate should eitherallow preventing combinations of potentially toxic drugs for tissuessuch as the muscle tissue, or defining combinations of drugs having acytotoxic effect useful for tumoral treatments. In human clinicalpractice, it has been frequently observed that the toxicity ofinhibitors of HMG CoA reductase may be exacerbated by certain drugcombinations. On the other hand, statins have been used for the past fewyears, for their cytotoxic properties in the treatment of certain formsof tumors (digestive tumors, breast tumors or lymphomas) and for theirpossible use as preventive agents (colon cancer or prostate cancer). Inthe large majority of the cases, in this latter indication, statins areused in combination with other anti-tumoral molecules.

With this in mind, the screen is made by using cells produced by themethod of the invention which are treated with inhibitors of thesynthesis of mevalonate such as inhibitors of HMG CoA reductase. Theconditions of the screen are produced as earlier and the cells aretreated with an inhibitor of HMG CoA reductase, simvastatin. Thecompounds are sorted according to their capability of enhancing thetoxic effects of simvastatin. By using this approach, about fifteencompounds were able to be selected in a bank of more than 1,200compounds. These are potential candidates for increasing the toxiceffects of this class of therapeutic molecules. Among these compounds,some of them are agents already known for their cell toxicity and othersare cytotoxic agents used in human clinical practice as anti-tumoralagents. On the other hand, scientific and clinical data indicate thatthere exists tumoral stem cells which are on the one hand responsiblefor resistance phenomena observed in conventional anti-tumoraltreatments. These tumoral <<stem cells>> are the result of a transitionfrom <<epithelial>> tissue to <<mesodermal>> tissue. The transitiontowards the mesodermis of tumoral epithelial cells gives these cells agrowth advantage and a larger capacity for forming tumors. Tumoral<<stem cells>> have certain features common to the differentiated<<MSC>> cells derived from pluripotent cells. Indeed, the <<MSC>> cellsare derived from pluripotent cells which are organized as an epitheliumfor evolving towards a mesodermal tissue. During the differentiation,they undergo a transition from the epithelial type to the mesodermaltype. Further, <<MSC>> cells express at a high level the marker CD44which is also expressed by tumoral <<stem>> cells. With these arguments,it is possible to believe that <<MSC>> cells derived from pluripotentcells represent a model of <<tumoral stem>> cells in vitro. Thedescribed screening then allows isolation of the compounds having aspecific activity against the tumoral <<stem>> cells. With the screeningsorting out the molecules which specifically enhance the toxicity ofstatins, it is possible to establish a rational strategy for definingthe molecules which have to be associated with statins for increasingtheir anti-tumoral potential and for targeting the <<tumoral stem>>cells.

The use of the cells produced by the method of the invention in thepresence or in the absence of an inhibitor of the synthesis ofmevalonate, in the presence or in the absence of mevalonate, allows highthroughput screening of the compounds for:

-   -   inhibiting the synthesis of mevalonate    -   attenuating the secondary effects of inhibitors of HMG CoA        reductase    -   preventing drug interactions having muscular toxicity effects    -   reducing muscle atrophy    -   potentializing cytotoxic effects inhibiting HMG CoA reductase        and defining drug combinations for their anti-tumoral activity        and in particular against tumoral <<stem>> cells.

The therapeutic applications of the thereby sorted out compounds arevery vast: hypercholesterolemias, muscle atrophies as well as tumoraldiseases.

Example 1 FGF2 Combined with Ascorbic Acid Improves the Robustness andEfficiency of the Techniques for Producing Differentiated MSC Cells orof the MSC Type Derived from Human Embryonic Stem Cells (hES) Having theCharacteristics of Multipotent Stroma Cells (MSC)

Introduction

Multipotent stroma cells or MSCs have been individualized, about tenyears ago. The ISCT (International Society for Cell Therapy) definesthese cells on several types of criteria:

-   -   Functional criteria: growth in a culture and substrates such as        culture plastic.    -   Identity criteria: absence of expression of CD45 and of CD34 and        expression of CD73, CD29 and CD44.    -   Differentiation potential in the osteogenic, chondrocyte and        adipose lineage.

In a first phase, the MSCs were isolated from bone marrow. Subsequently,these cells were partly purified from many tissues such as adiposetissue, peripheral blood, liver, lungs, muscles, placenta, amnioticliquid or umbilical cord blood. MSCs are involved in tissue repairincluding bone repair, in immunomodulation, in the control of angiogenicresponse and in the tumoral phenomenon.

These features make MSCs indispensable cell tools for toxicologicalstudies within the musculo-skeletal sphere and for discovering newtherapeutic routes in different fields (orthopedics, vascular diseases,inflammatory diseases, cancer and gene transfer). The isolation andproduction of stem cells from human tissues however have manylimitations:

-   -   Accessibility of human tissue samples    -   Isolation and production conditions which have to be adapted for        each of the tissues.    -   Limited growth capacity.    -   Phenotype instability    -   Interindividual variability

The existence of human pluripotent stem cells gives the possibility ofaccessing normal human cells without any limitations. There exist twotypes of pluripotent cells: embryonic stem cells (hES) and induced stemcells (iPS). The essential features of pluripotent cells are thecapability of self-renewal without any limitations and the possibilityof differentiation into all the cell types making up an entire organism.

The possibility since the work of J. Thompson (Thompson et al. 1998) ofusing human embryonic stem cells (hES) has opened very manypossibilities, some of which may have clinical implications. Bothessential features which are the capability of self-renewal without anylimitations and the capability of differentiation into all the celltypes making up an adult organism makes these cells an extremely usefultool. Experimentally, human embryonic stem cells have alloweddevelopment of differentiation procedures and isolation of many types ofcells (cardiac cells, smooth muscle cells, neural cells, keratinocytes,hematopoietic cells, insulin-producing cells).

A) Description of the Procedures for Isolating and Producing Cells ofthe MSC Type Having Features Common to MSCs Derived from Human EmbryonicStem Cells (hES).

The cells are produced from non-differentiated hES cells.

The production conditions are the following. The islets ofnon-differentiated cells which may be identified by their morphologicalcharacteristics are the basis of the cell material for producing thesecells.

-   -   1) Mechanical transplantation of the islets recognized by the        morphology of hES cells.        -   a. Change the hES cell dishes for EB medium (see Table 1);        -   b. Striate with a needle and then detach the aggregates. The            29 G needle is mounted on a 1 mL syringe. Select the            colonies without any sign of morphological differentiation.            Several tens of colonies are thereby isolated. The colonies            are counted in the presence of trypan blue for appreciating            their viability;        -   c. Centrifugation at 900 rpm for 1 minute for forming            pellets without crushing them, in 15 mL tubes;        -   d. Take up the pellet into 1 mL of EB medium. Suck up,            discharge for partly dissociating the cell aggregates. The            size of the aggregates should be of several tens of cells;        -   e. Trypan blue counting: determination of the number of            aggregates and viability.    -   2) Selection and amplification of MSC cells:        -   f. A specific number of aggregates (several tens of them) is            sown in the EB medium on gelatinized culture dishes. 50 to            200 aggregates are sown per 25 cm² flasks;        -   g. The first change is carried out after 48 hours and then            every 2 to 3 days. Two culture conditions are tested. Under            the first condition, the source of growth factors is limited            to fetal calf serum (EB medium). For the second, FGF2 (10            ng/ml) and 1 mm of ascorbic acid 2-phosphate are added to            the fetal calf serum. This medium is called EBMOD.        -   h. After 4 to 5 days, the cells emerge from the aggregates,            bind to the substrate and start to divide.        -   i. Between day 10 and day 15, the cells are transplanted by            using a solution of trypsin combined with EDTA.        -   j. After washing with culture medium by centrifugation and            counting, the cells are sown on gelatinized supports with a            density ranging from 2,000 cells to 10,000 cells per cm².        -   k. The first transplantations are carried out between day 5            and 10.

Within the scope of this procedure, we compared two culture media: themedium which we called EB and a medium EBMod to which we added FGF2 at aconcentration of 10 μg/ml and Ascorbic Acid 2-Phosphate (AA2P) at theconcentration of 1 mM. The composition of the EB medium is indicated inTable 1.

TABLE 1 Composition of the EB medium Stock Final Volume concentrationconcentration (total of 500 ml) KO-DMEM sfc 500 ml 390 ml FCS (Fetalcalf serum) 100% 20% 100 ml β-mercaptoethanol 50 mM 50 μM 500 μlGlutamax 200 mM 2 mM 5 ml NEAA 100X 1X 5 ml (MEM aa not essential)Penicillin/Streptomycin 10000 IU/ml 10 IU/ml 500 μl

For this study, hES embryonic stem cells WT4 (KCL-002), XY were used.This hES cell line was isolated by Stephen Minger from the WolfsonCentre for Age-Related Diseases, King's College London. It stems fromthe UK Stem Cell Bank: UK Stem Cell Bank, National Institute forBiological Standards and Control. Similar results are obtained with SA01embryonic stem cells. This procedure is also applicable to embryonicstem cells which have mutations which represent pathological models.

B) Production of Differentiated Cells of the MSC Type SharingCharacteristics of Multipotent Stroma Cells (MSC) Derived from HumanEmbryonic Stem Cells (hES).

The two conditions of media were analyzed on the production and thecharacterization of cells obtained according to the procedure describedearlier. In this case, the differentiated cells of the MSC type werederived from WT4 human embryonic stem cells. The results are indicatedin the two following tables.

TABLE 2 Results of a growth curve obtained in the absence of FGF2 andAA2P MSWT4 Number of cumulated divisions Number of accumulated cells D 0D 10 ND 7.7 10⁵ D 17 ND 6.6 10⁵ D 24 1.15 1.7 10⁶ D 30 4.02 1.2 10⁷ D 386.18 5.6 10⁷ D 43 7.86 1.8 10⁸ D 76 6.54 7.2 10⁷

TABLE 3 Results obtained in the presence of FGF2 and AA2P MSC WT 4Number of Number of FGF2 AA2P cumulated divisions cumulated cells D 0 D10 ND 1.5 10⁶  D 17 2.96 1.2 10⁷  D 21 4.54 3.6 10⁷  D 23 6.1  1 10⁸ D30 9.6 1.2 10⁹  D 38 13.3 1.6 10¹⁰ D 43 16.7 1.6 10¹¹ D 48 21.3  4 10¹²D 52 24.8 4.5 10¹³ D 59 30.6 2.5 10¹⁵ D 65 36  1 10¹⁷ D 71 40 1.8 10¹⁸ D78 43 1.4 10¹⁹ D 87 44.5 3.8 10¹⁹ D 94 45.5 7.6 10¹⁹ D 101 48.6 6.5 10²⁰D 118 50.2 1.9 10²¹

Under both medium conditions, it is possible to derive adhering cellswhich are capable of multiplying under both culture conditions. In theabsence of FGF and of ascorbic acid 2-phosphate, the number of isolatedcells is much less significant. These cells after a few cell divisionsbecome senescent and are incapable of being efficiently amplified underthese conditions. The amount of cells obtained is more substantial inthe presence of the medium containing FGF and ascorbic acid 2-phosphate.Under the latter conditions, the number of cells may be increased byseveral log factors. The introduction of this modification also allowsshortening of the production times for cells of the MSC type. Underthese conditions, from about 100 aggregates stemming from humanembryonic stem cells, it is possible to produce 10⁸ cells within 23 daysof cultures which have a significant replicative potential (more thanabout thirty cell divisions). It should be noted that thesemodifications do not alter the capability of these cells of entering asenescence phase, indicating the non-transformed nature of the therebyproduced cells. It should be noted that under these conditions, it ispossible to produce MSC cells with all human normal embryonic stem cellsor having mutations. This method was applied successfully for embryonicstem cells from embryos affected with Steinert's disease. Theseconditions give the possibility of increasing the robustness of themethod for producing differentiated cells of the MSC type derived fromhuman embryonic stem cells.

C) Characterization of Differentiated Cells of the MSC Type Derived fromHuman Embryonic Stem Cells (hES) by Flow Cytometry.

The characterization of differentiated cells produced by the productionmethod from human embryonic stem cells (hES) was carried out by flowcytometry. This common technology allows qualitative and quantitativeanalyses of the molecules present at the surface of the cells by usingspecific antibodies. For this step, antibodies directed against themembrane markers associated with totipotency conditions and againstmarkers of MSC cells were used. The thereby produced cells do notexpress at their surfaces the antigen TRA 1-60 which is associated withtotipotency conditions. Similar results are obtained with antibodiesdirected against other markers of totipotency such as SSEA3 and TRA1-80. These are therefore cells having lost their specific pluripotentnature of embryonic stem cells. On the other hand, these cells expressmarkers associated with MSC cells such as CD73, CD29, CD44, CD166 andCD105. The results obtained are shown in Table 4.

TABLE 4 Flow cytometry analysis of membrane markers of MSC cellsAntibody Fluorescence intensity % of positive cells CD73 400 99% CD29672 100%  CD44 624 100%  CD166 145 98% CD105 283 95% SSEA-1 567  1%

The cells produced by the production method are homogeneous to more than95% for the markers CD73, CD29, CD44, CD166 and CD105. These results arenot modified by the presence of FGF2 and AA2P. The thereby producedcells have the main characteristics of MSC cells for their surfacemarkers.

D) the Combined Presence of FGF2 and Ascorbic Acid 2-Phosphate in theCulture Medium Increases the Growth Capacity and the Expression of SSEA4in Cells Produced by the Method of the Invention.

The role of FGF2 and of ascorbic acid 2-phosphate on the growthcapacities of the isolated cells according to the method of theinvention was analyzed on cells derived from human embryonic stem cellsSA01. The cells are cultivated in the presence of 20% fetal calf serum(control condition) and in the presence or in the absence of FGF2 and ofascorbic acid 2-phosphate (AA2P). The results which represent themaximum number of cells obtained before entering senescence areindicated in the following Table 5.

TABLE 5 Control Control Control (FCS20%) (FCS20%) (FCS20%) FGF2 ControlFGF2 AA2P 10 ng/ml + (FCS20%) 10 ng/ml 1 mM AA2P 1 mM Maximum 7. 10⁸ 4.710¹³ 3.7 10¹⁵ 1.5 10²⁰ number of cells before senescence

The combined presence of FGF2 and AA2P gives the possibility ofincreasing by about a log factor of 12, the amount of accumulatedmaximum cells. FGF2 and ascorbic acid individually have a much smallereffect on the increase in the amount of cells, a little less than 5 logfactors for FGF2 and a little less than 6 log factors for ascorbic acid2-phosphate. The effect of FGF2 and of the ascorbic acid is clearlysynergistic on the maximum cell amount of accumulated cells.

SSEA4, a membrane marker, is expressed on the pluripotent cells and on acertain number of progenitor cells from different tissues such asmarrow, adipose tissue or neural tissue.

The presence of SSEA4 on cells cultivated under these differentconditions were analyzed by flow cytometry by using a specific antibodydirected against SSEA4. The results are indicated in the following Table6:

TABLE 6 Control Control Control (FCS20%) (FCS20%) (FCS20%) FGF2 ControlFGF2 AA2P 10 ng/ml + (FCS20%) 10 ng/ml 1 mM AA2P 1 mM Average 7 10 7 26fluorescence intensity

Neither FGF2 nor ascorbic acid 2-phosphate significantly increase theexpression of SSEA4. On the other hand, the combination of bothmolecules increases the amount of SSEA4 by a factor of 3.7. Thisfluorescence level is comparable with the one observed with humanpluripotent cells. The combined presence of FGF2 and ascorbic acid2-phosphate increased in a combined way the maximum number ofaccumulated cells and the expression level of SSEA4.

E) the Cells of the MSC Type Produced According to the Method of theInvention have a Normal Karyotype.

The karyotype of <<MSC>> cells was analyzed on chromosomes in ametaphase and proved to be without any detected abnormality in thepresence and in absence of FGF2 and AA2P.

F) the MSC Type Cells Produced According to the Method of the Inventionhave Osteogenic Potential.

In an osteogenic medium, cells of the MSC type produced according to themethod of the invention are capable of expressing functions of bonetissue. After 14 days of cultivation in a medium comprisingdexamethasone, B glycerophosphate, the cells of the MSC type producedaccording to the method of the invention express alkaline phosphataseand form mineralization nodules. The mineralization nodules are revealedby a specific histochemical technique, Von-Kossa's staining.

G) Freezing Differentiated Cells of the MSC Type Derived from HumanEmbryonic Stem Cells (hES).

The thereby produced cells may be kept by freezing them. The freezingmedium is the following: 90% of serum and 10% of DMSO. Freezing of thecells produced in the medium in the presence of FGF2 and AA2P waspracticed at passage 7 and at a concentration of 10⁶ per ml.

The MSC cells produced under both conditions are very efficientlypreserved by freezing. The freezing does not change the growthcharacteristics of MSC cells. These freezing techniques give thepossibility of storing a large number of cells without modifying theirbiological characteristics.

H) Conclusions

The presence in the medium for amplifying differentiated MSC cellsderived from hES cells with FGF2 and ascorbic acid 2-phosphate gives thepossibility of:

-   -   increasing the robustness of the techniques for producing the        differentiated cells from embryonic stem cells    -   accelerating the production of these cells    -   increasing the maximum number of produced cells    -   increasing the proliferation potential    -   obtaining cells having a normal karyotype    -   specifically expressing SSEA4 as a marker of totipotent cells        and of progenitor cells

The cells produced in the medium in the presence of FGF2 and ascorbicacid 2-phosphate have characteristics associated with MSC cells:

-   -   Adhesion to the substrates    -   High but finite growth potential    -   A karyotype without any detectable abnormality.    -   Preservation by freezing    -   Homogeneity of expression for the markers CD73, CD29, CD44,        CD166, CD105.    -   Osteogenic potential

Example 2 FGF2 Associated with Ascorbic Acid Improves the Robustness andthe Efficiency of the Techniques for Producing Differentiated Cells ofthe MSC Type Derived from Human Pluripotent Cells (iPS) HavingCharacteristics of MSC Cells

A) Description of the Procedures for Isolating and Producing CellsHaving Characteristics Common with Those of MSCs from InducedPluripotent Human Cells (IPS).

The iPS cells iPS4603Cl5 and iPS4603PClA were produced from humanprimary fibroblasts by using Yamanaka's procedure which introduces thefollowing genes: Oct4, Sox2, cMyc and Klf 4. The gene transfer wascarried out with retroviruses of the Moloney type. Viral transductionwas carried out by co-infection with the 4 viruses bearing each of these4 genes. Treatment by valproic acid allowed an increase in theefficiency of the reprogramming. After transduction, the cells arecultivated in the presence of nutritive tissue consisting of mouseembryonic fibroblasts in a calcium medium promoting emergence of humanpluripotent cells. The essential constituent of the specific medium forgrowing pluripotent human cells is FGF2. The medium is changed every 24hours. After 2 to 3 weeks, the first colonies having the characteristicsof pluripotent cells are visible. These colonies are then isolated andmechanically transplanted. Two populations are then built up: amonoclonal population (iPS iPS4603Cl5) and a polyclonal population(iPS4603PClA). The thereby isolated cells have the followingcharacteristics:

-   -   Extinction of the transduced exogenous genes used for        reprogramming.    -   Expression of totipotency markers: SSEA4 and TRA-1-81, TRA 1-60    -   Normal karyotype    -   Growth capacity while maintaining the pluripotency condition in        the medium in the presence of FGF2    -   Differentiation capacity after forming embryonic bodies        (ectodermis, endodermis, mesodermis)

These cells have characteristics of induced pluripotent cells (iPS).These cells were therefore used for producing cells of the MSC type.

The production conditions are the following. The islets ofnon-differentiated cells which may be identified by their morphologicalcharacteristics are the basis of the cell material for producing thesecells.

-   -   1) Mechanical transplantation of islets recognized by the        morphology of hES cells        -   l. Change the iPS cell dishes for EB medium (see Table. 1).        -   m. Striate with a needle and then detach the aggregates. The            29 G needle is mounted on a 1 ml syringe. Select the            colonies without any signs of morphological differentiation.            Several tens of colonies are thereby isolated. The colonies            are counted in the presence of trypan blue for appreciating            their viability.        -   n. Centrifugation at 900 rpm for 1 minute in order to form            pellets without crushing them, in 15 ml tubes        -   o. Take up the pellet into 1 ml of EB medium. Suck up,            discharge, for partly dissociating the aggregates of cells.            The size of the aggregates should be of a few tens of cells        -   p. Trypan blue counting: Determination of the number of            aggregates and viability.    -   2) Selection and amplification of cells of the MSC type        -   q. A specific number of aggregates (several tens of them) is            sown in the EB medium on gelatinized culture dishes. 50 to            200 aggregates are sown per 25 cm² flask.        -   r. The first change is carried out after 48 hours and then            every 2 to 3 days. Two culture conditions are tested. Under            the first condition, the growth factor source is limited to            fetal calf serum (EB medium). For the second condition, FGF2            (10 ng/ml) and 1 mM of ascorbic acid 2-phosphate are added            to the fetal calf serum. This medium is called EBMOD        -   s. After 4 to 5 days, the cells emerge from the aggregates,            bind to the substrate and start to divide.        -   t. Between day 10 and 15, the cells are transplanted by            using a solution of trypsin combined with EDTA.        -   u. After washing with the culture medium by centrifugation            and counting, the cells are sown on gelatinized supports            with a density ranging from 2,000 cells to 10,000 cells per            cm².        -   v. The first transplantations are carried out between day 5            and 10.

Within the scope of this procedure, we compare two culture mediums: themedium which we called EB and a medium EBMod to which FGF2 was added ata concentration of 10 μg/ml and ascorbic acid 2-phosphate at aconcentration of 1 mM. The composition of the EB medium is indicated inTable 1.

TABLE 7 Composition of the EB medium Stock Final Total of concentrationconcentration 500 ml KO-DMEM qsp 500 ml 390 ml FCS (Fetal calf serum)100% 20% 100 ml β-mercaptoethanol 50 mM 50 μM 500 μl Glutamax 200 mM 2mM 5 ml NEAA 100X 1X 5 ml (MEM aa not essential)Penicillin/Streptomycine 10000 IU/ml 10 IU/ml 500 μlB) Production of Differentiated Cells Showing Characteristics ofMultipotent Stroma Cells (MSC) from Induced Pluripotent Human Cells(iPS).

Both medium conditions were analyzed on the production andcharacterization of the cells obtained by following the procedureidentical with the one described in Example 1

From the tenth day, the number of cells observed is larger in the mediumin the presence of FGF and ascorbic acid 2-phosphate. This trend isconfirmed during cultivation. The obtained results are indicated in thefollowing table.

TABLE 8 Growth curve in the absence of FGF2 and AA2P Number of Number ofMSC 4603 Cl 5 cumulated divisions cumulated cells D 0 ND ND D 10 ND ND D20 2.2 1.1 10⁶  D 24 4.5 5.3 10⁶  D 29 7.6 4.8 10⁷  D 38 12.1  1. 10⁹ D45 15.9 1.4 10¹⁰ D. 52 18.2 7.4 10¹⁰ D 57 20.1 2.8 10¹¹ D 66 22.2 1.110¹² D 80 23.9 3.7 10¹² D 87 25.4  1 10¹³

TABLE 9 Growth curve in the presence of FGF2 and AA2P MSC 4603 Cl Numberof Number of 5 FGF2 AA2P cumulated divisions cumulated divisions D 0 NDND D 10 ND ND D 13 0.3 9.7 10⁵  D 17 2.4 4.2 10⁶  D 22 7.1 1.1 10⁸  D 2711 1.6 10⁹  D 31 15 2.5 10¹⁰ D 38 20.6 1.2 10¹² D 44 25.9 4.8 10¹³ D 5031 1.7 10 ¹⁵ D 55 35.9  5 10¹⁶ D 60 38.7 3.6 10¹⁷ D 64 42.3 4.2 10¹⁸ D71 47.3 1.3 10²⁰ D 78 51.7 2.9 10²¹ D 85 56.3 6.7 10²² D 92 59.7 7.110²³

Under both medium conditions, it is possible to derive adhering cellswhich are capable of multiplying under both cultivation conditions.However it should be noted that the amount of cells obtained is largerin the presence of the medium containing FGF and ascorbic acid2-phosphate. Under the latter conditions, the number of cells may beincreased by several log factors. The introduction of this modificationalso allows shortening of the times for producing cells of the MSC type.Under the latter conditions, from about 100 aggregates stemming fromhuman embryonic stem cells, it is possible to produce 10⁸ cells within22 days of cultivation which have significant replicative potential (ofmore than about thirty cell divisions).

In the absence of FGF2 and of ascorbic acid 2-phosphate, the number ofisolated cells is much less significant. After three weeks ofcultivation, the number of cells obtained is twenty times lesssignificant. After 50 days of cultivation, the difference is 10⁵. Afterabout twenty cell divisions, the cells produced under these conditionsbecome senescent.

These results obtained with a clone of iPS cells are confirmed with apolyclonal population of iPS cells. The results obtained with apolyclonal population are similar.

C) Freezing Differentiated Cells Sharing the Characteristics of Cells ofthe MSC Type Derived from Induced Pluripotent Human Cells (IPS).

The thereby produced cells may be preserved by freezing. The freezingmedium is the following: 90% of serum and 10% of DMSO. Freezing of thecells produced in an EBMod medium was practiced in passage 7 and at theconcentration of 10⁶ per ml. The viability is measured by an exclusiontest with trypan blue.

The MSC cells produced under both conditions are very efficientlypreserved by freezing. Freezing does not modify the growthcharacteristics of the MSC cells. These freezing techniques give thepossibility of storing large numbers of cells without modifying theirbiological characteristics.

D) MSC Cells Produced According to the Method of the Invention haveNormal Karyotype.

The karyotype of MSC cells was analyzed on chromosomes in a metaphaseand proved to be without any detectable abnormality in the presence andin the absence of FGF2 and AA2P.

E) Characterization of Differentiated Cells Sharing the Characteristicsof Multipotent Stroma Cells <<MSC>> from Induced Pluripotent Human Cells(iPS) by Flow Cytometry.

The characterization of the differentiated cells produced by theproduction method from human embryonic stem cells (hES) was carried outby flow cytometry. This current technology allows qualitative andquantitative analyses of the molecules present at the surface of thecells by using specific antibodies. For this step, antibodies directedagainst markers of membranes associated with the totipotency conditionand against markers of MSC cells were used. The thereby produced cellsdo not express at their surfaces, the antigen TRA 1-60 which isassociated with the totipotency condition. The other totipotency markerslike SSEA3 and TRA 1-80 give similar results. These are therefore cellshaving lost the specific pluripotent nature of the embryonic stem cells.On the other hand, these cells express markers associated with MSC cellssuch as CD73, CD29, CD44, CD166 and CD 105.

The obtained results are shown in Table 10.

TABLE 10 Flow cytometry analysis of membrane markers of MSC cellsAntibody Fluorescence intensity % of positive cells CD73 490 93% CD29742 93% CD44 897 99% CD166 228 97% CD105 198 98% SSEA-1 326  1%

The cells produced by the production method are homogeneous to more than93% for the markers CD73, CD29, CD44, CD166 and CD105. The therebyproduced cells have the main characteristics of MSC cells for theirsurface markers.

Identical results are obtained with populations of polyclonal iPS cells.The thereby described procedure gives the possibility, with similarresults, of obtaining differentiated MSC cells by using iPS cellsobtained with the technique described by Thompson. The efficiency of theprocedure described does therefore not depend on reprogrammingtechniques.

F) the Combined Presence of FGF2 and Ascorbic Acid 2-Phosphate in theCulture Medium Increases the Growth Capacity and the Expression of SSEA4by the Cells Produced by the Method of the Invention.

SSEA4, a membrane marker is expressed on pluripotent cells and on acertain number of progenitor cells from different tissues such asmarrow, adipose tissue or neural tissue.

The presence of SSEA4 on cells cultivated under these differentconditions was analyzed by flow cytometry by using a specific antibodydirected against SSEA4. The results obtained with cells derived frompluripotent cells induced from human fibroblasts (iPS iPS4603Cl5) areindicated in the following table:

TABLE 11 4 % of positive cells Average fluorescence Control FCS 20%35.6% 132 Control FCS 20% + FGF2 79.6% 336 10 ng/ml + AA2P 1 mM

The presence of the combination of FGF2 and ascorbic acid 2-phosphateincreases the number of cells expressing SSEA4 and the averagefluorescence intensity.

The combined presence of FGF2 and ascorbic acid 2-phosphate increasestogether the maximum number of accumulated cells and the expressionlevel of SSEA4.

G) the Cells of the MSC Type Produced According to the Method of theInvention have Osteogenic Potential.

In an osteogenic medium, the cells of the MSC type produced according tothe method of the invention are capable of expressing functions of bonetissue. After 14 days of cultivation in a medium comprisingdexamethasone, B glycerophosphate, the cells of MSC type producedaccording to the method of the invention express alkaline phosphataseand form mineralization nodules. The mineralization nodules are revealedby a specific histochemical technique: Von Kossa's staining.

H) Conclusions

The presence of FGF2 and of ascorbic acid 2-phosphate in the medium foramplifying the cells gives the possibility of:

-   -   Increasing the robustness of the techniques for producing        differentiated cells from induced pluripotent cells (iPS)    -   Accelerating the production of these cells    -   Increasing the number of produced cells    -   Increasing the proliferation potential without modifying their        capability of senescence.    -   Obtaining cells having a normal karyotype    -   Specific expression of SSEA4, a marker of totipotent cells and        of progenitor cells.

The cells produced in the medium and in the presence of FGF2 andascorbic acid 2-phosphate have the characteristics associated with MSCcells:

-   -   Adhesion to the substrates    -   High but finite growth potential.    -   Preservation by freezing    -   Homogeneity of expression for the markers CD73, CD29, CD44 and        CD166.    -   Osteogenic potential

Example 3 Differentiated Cells of the MSC Type Derived from HumanEmbryonic Stem Cells (hES), Tools for Predictive Toxicology of MuscularTissue for High Throughput Screening Techniques

A) Human Muscle Precursor Cells (MPC): Tools for Predictive Toxicology.

Human muscle precursor cells (MPC) are good indicators of musculartoxicity. Indeed, the cell growth of MPCs is inhibited by the presenceof statin in the growth medium. This inhibition is dose-dependent. Thepresence of mevalonate allows the inhibition to be totally raised. Thisinhibition is therefore accomplished through the inhibition of HMG CoAreductase, an enzyme responsible for the production of mevalonate, aprecursor in the synthesis of cholesterol. With this cell test system,the toxicity of different statins, having been used in human clinicalpractice, has been tested. The obtained results are indicated in thepatent: culture composition, culture method and their uses in PCT WO2004/0055174 A1.

At a concentration of 1 μM, the whole of the statins have toxicity formuscle cells. This toxicity is also dependent on the type of statin.Cerivistatin has a greater toxicity at all the tested concentrations.These results are in good correlation with clinical observations whichhave shown a significant toxicity of cerivistatin which was withdrawnfrom the market for its significant muscular toxicity. This cell test(bioassay) using MPCs therefore has a predictive value for musculartoxicology. However, there exists intrinsic limitations to this type ofbioassay which are related to the biological characteristics of MPCcells. MPC cells are human primary cells isolated from muscle tissuewhich have variations from batch to batch, limited replicationcapacities and phenotype instability during the passages.

B) Differentiated Cells of the MSC Type Derived from Human EmbryonicStem Cells as Tools for Predictive Toxicology.

The goal is therefore to replace MPC cells with cells which may beproduced in a reproducible way in a large amount and having sensitivityto inhibitors of HMG CoA reductase. Differentiated cells of the MSC typederived from pluripotent human cells (hES and iPS) may be producedwithout any limitations on number (see Example 1). Table 2 indicatesthat these cells have sensitivity to statins close to the one observedwith MPC cells. Differentiated cells of the MSC type derived from hEScells (SA01) designated as: <<MSC>> SAMU 43, are cultivated withincreasing doses of Mevolin (Lovostatin) and of Simvastatin in thepresence and absence of mevalonate. After 7 days of cultivation, thecells are set, stained and then counted by image analysis. The obtainedresults are shown in the following table:

TABLE 12 Dose response curves of << MSC >> SAMU43: effect of mevalonateConcentration in M 0 5 10⁻⁶ 10⁻⁶ 5 10⁻⁷ 10⁻⁷ 5 10⁻⁸ Mevinolin 9842 1223232 6934 10021 10167 Simvastatin 9553 45 1750 5938 10215 9467Mevinolin + 8223 8148 9525 9003 9423 9270 Mevalonate Simvastatin + 96999281 8991 9310 8834 8408 Mevalonate

The <<MSC>> SAMU43 cells are sensitive to statins in a dose-dependentway. Like for MPCs, the presence of mevalonate prevents toxicity of thestatins. The observed toxicity is therefore due to the inhibition of thesynthesis of mevalonate, a precursor of cholesterol. It is thereforepossible to use differentiated cells of the MSC type derived fromembryonic stem cells for evaluating cell toxicity of the inhibitors ofthe synthesis of mevalonate. These differentiated cells of the MSC typederived from embryonic stem cells are therefore an unlimited source forthis type of cell test.

C) Differentiated Cells of the MSC Type Derived from Embryonic StemCells: Tools for Screening.

The development of high throughput screening technology requires thepossibility of cultivating the cells in multiwells without altering thebiological functions and having automatable read-out systems. With ATPdosage, it should be possible to determine the number of cellsautomatically.

In this test, the light emission is proportional to the amount of cellATP. The following experiments allowed verification of the relationshipbetween the amount of ATP and the number of cells. Increasing numbers ofcells were either analyzed by image analysis or by determining theamount of ATP. The cells were cultivated in 96-well multiwells. From 10²cell to 10⁴ cells were analyzed by image analysis and by dosage of theATP amounts.

There exists a good linearity between the number of cells determined bythe image analysis and the amount of detected ATP by luminescence withan excellent R² of 0.9831. Determination of the number of cells by theATP amount is therefore possible. This approach allows automated readoutof the number of cells. Similar results are obtained by using traces ofmitochondrial activity. Differentiated cells of the MSC type, derivedfrom hES cells, <<MSC>> WT, were sown on 384 multiwells gelatinizedbeforehand by using a distribution robot of the Bravo type, at theconcentration of 2,000 cells per well. The amount of cells was evaluatedby Cell Titer Glow after 72 hours of cultivation in the presence or inthe absence of simvastatin in a culture medium containing 20% of fetalcalf serum. The obtained results are indicated in Table 4.

TABLE 13 Toxicity of simvastatin for MSCs derived from embryonic stemcells in 384-well multiwells. DMSO CV DMSO Statin CV Statin 2 DMSO/ 0.2%0.2% 2 μM 2 μM Statin

 MSC 

987206 5.4 474583 15.9 2.08 WT CV means coefficient of variation, whichis the standard deviation divided by the average.

Simvastatin is toxic for differentiated cells at 2 μM with a <<DMSO overstatin>> ratio of more than 2. The coefficients of variation are notvery high, between 2.9 and 15.9.

Toxicity is not limited to simvastatin, but is also observed with twoother statins. In all the cases, this toxicity is abolished bymevalonate indicating that this inhibition is accomplished so that theinhibition of HMG CoA reductase, an enzyme responsible for the synthesisof mevalonate. For this experiment, the differentiated <<MSC>> SA01cells derived from hES cells (SA01) were tested in 384-well plates byusing a distribution robot and cell viability was measured by Cell TiterGlow after 72 hours of cultivation. The obtained results are in Table 5and are expressed as a percentage of the obtained result without anytreatment.

TABLE 14 Comparative toxicity of statins on differentiated cells 

 MSC 

 SA01 derived from embryonic stem cells. Simvastatin LovostatinFluvostatin Without Mevalonate 48.7% 53.6% 32.8% With Mevalonate 98.3%96.4%  104%

The three tested statins have toxicity for these cells and in all thecases, the presence of mevalonate abolishes this toxicity. It shouldalso be noted that fluvastatin is more toxic than simvastatin and thatthe latter is more toxic than lovostatin. These results are very similarto those obtained with the MPCs.

In order to experimentally validate the <<MSC>> cells derived from hEScells, we screened a bank of small molecules, the Prestwick bank. Thisbank consists of more than 1,200 molecules which represent the largemajority of the products used in human clinical practice. The substancesare used at the concentration of 2.5 μM. The cells are sown in 96-wellor 384-well multiplates gelatinized beforehand in the presence or in theabsence of mevalonate at the concentration of 2 mM. 24 hours later, thecompounds of the bank are distributed by means of a robot of theVelocity type. The amount of ATP per well is determined from a CellTiter Glow test 72 after adding the compounds from the bank. Thescreening criteria were the following:

-   -   A toxicity of more than 35% in the absence of mevalonate    -   A toxicity of less than 20% in the presence of mevalonate

Three different screening tests were carried out. For the firstscreening, the MSC cells were derived from hES human cells SA01. Thetest was carried out in 28 multiwells with 96 wells in the presence of aculture medium containing 20% fetal calf serum. The average of the Zfactors was 0.47. Under these conditions, 11 molecules over more than1,200 tested were able to be isolated, including the three statinspresent in the bank (Lovostatin, Simvastatin, Fluvastatin).

The second screening was carried out by using MSC cells derived fromanother line of human embryonic stem cells, the line VUB01. In thistest, the cells were sown in 384-well multiplates. The conditions of thescreening were produced like the previous one. The average Z′ factor was0.55. The number of molecules sorted out in this way was 15 includingthe three statins present in the bank. A third screening was carried outon a 384-well plate with the same cells as those of the first screening.In this case, the Z′ factor was 0.59. The number of molecules sorted outin this way was three including the three statins contained in the bank.

The obtained results in the three independent screenings are illustratedand summarized in the following table.

TABLE 15 Z′ Number of Cells Format Factor Hits Statin Screening 1

 MSC 

 SA001 96 0.47 11 3 Screening 2

 MSC 

 VUB001 384 0.54 15 3 Screening 3

 MSC 

 SA001 384 0.59 3 3

The only molecules which we again found at each screening were the threestatins contained in the bank. Therefore the conclusion may be drawnthat the conditions of this bioassay using <<MSC>> cells derived fromhuman embryonic stem cells, give the possibility of screening moleculesinhibiting the synthesis of mevalonate in a sensitive and robust way.MSC cells derived from embryonic stem cells are excellent tools for highthroughput screening. These properties are observed with all the MSCsderived from hES cells.

D) Conclusions

The differentiated cells of the MSC type derived from human embryonicstem cells have a sensitivity to statins of the same type as MPC cellsand are robust, sensitive, specific tools and suitable for highthroughput techniques for screening products having toxicity for muscletissue and for inhibitors of the synthesis of mevalonate.

Example 4 Differentiated Cells of the MSC Type Derived from HumanInduced Pluripotent Cells (iPS) as Tools for Predictive Toxicology ofMuscle Tissue for High Throughput Screening Techniques

Induced pluripotent cells iPS which share the essential characteristicsof embryonic stem cells (self-renewal without any limitations and thepossibility of differentiation into all the cell types making up anentire organism) are both easier to produce and pose less regulatoryquestions. These properties make these cells and their derivatives, goodcandidates for high throughput screening techniques.

A) Differentiated MSC Cells Derived from Human Induced Pluripotent StemCells: Tools for Predictive Toxicology in High Throughput Screening.

Development of high throughput screening technology requires thepossibility of cultivating cells in multiwells without altering thebiological functions and having automatable readout systems. The dosageof ATP should be able to determine the number of cells automatically.

In this test, the light emission is proportional to the amount of cellATP. The preliminary experiments gave the possibility of verifying therelationship between the amount of ATP and the number of cells. In thistype of test, the measured amount of ATP represents the number of cells.

Differentiated <<MSC>> cells derived from human induced pluripotent stemcells iPS were sown at a density of 2,000 cells per well among 384 wellsand cultivated for 72 hours in the presence and in the absence ofSimvastatin at the concentration of 2 μM. The different followingculture conditions were tested:

-   -   Culture medium and ascorbic acid 2-phosphate    -   Culture medium and 1% of fetal calf serum    -   Culture medium and 10% of fetal calf serum    -   Culture medium and 20% of fetal calf serum

The goal of the experiment was to both test the robustness of the testand to define the optimum conditions for conducting this test.

The results in figures are shown in the following table:

TABLE 16 Simvastatin CV DMSO CV Inhibition % Z′ Factor AA2P 235 856 5.6  631 378 3 63 0.76 SVF 1%  53 212 12.2   747 366 6 93 0.78 SVF 10% 294847 14.6 1 343 308 6.3 78 0.63 SVF 20% 471 949 14.4 1 411 750 7 67 0.76CV means coefficient of variation, being the standard deviation dividedby the mean. AA2P = Medium + 1 mM AA2P SVF 1% = Medium + 1% of fetalcalf serum SVF 10% = Medium + 10% of fetal calf serum SVF 20% = Medium +20% of fetal calf serum

Under all the tested conditions, simvastatin has a large toxic effect onthese cells, of more than 60%. This toxicity is very similar to the oneobserved with muscle precursor cells (MPCs). It should also be notedthat under all the experimental conditions, the Z′ factor which is thestatistical indicator of the size effect is greater than 0.60 whichindicates that these tests may be used for high throughput screenings.On the other hand, it is possible to practice this type of test in asynthetic medium without any presence of animal serum. In this case, theobserved toxicity is 63% with a Z′ factor of 0.76. The optimum cultureconditions for this test are the medium in the presence of 1% of fetalcalf serum. Under these conditions, the reduced number of cells is 93%with a Z′ Factor of 0.78.

In order to experimentally validate the <<MSC>> cells derived from iPScells, we screened a bank of small molecules, the Prestwick bank. Thisbank consists of more than 1,200 molecules which represent the largemajority of products used in human clinical practice. The substances areused at the concentration of 2.5 μM. A schematic illustration of theprocedure is shown in Annex 1. The cells are sown in 96-well or 384-wellmulti-plates gelatinized beforehand in the presence and in the absenceof mevalonate at the concentration of 2 mM. 24 hours later, thecompounds of the bank are distributed by means of a robot of theVelocity type. The amount of ATP per well is determined by means of aCell Titer Glow test 72 after adding the compounds of the bank. Thescreening criteria were the following:

-   -   Toxicity of more than 80% in the absence of mevalonate    -   Toxicity of less than 50% in the presence of mevalonate

The screening test was carried out by using <<MSC>> cells derived fromiPS cells. 2,000 cells were sown in 16 plates of 384 wells in thepresence and in the absence of mevalonate. 24 hours later, the cellswere put into contact with the molecules from the Prestwick bank. Themeasurement was conducted after 72 hours of cultivation. The Z′ factorof this test is greater than 0.70.

With these criteria, we only identify three molecules which are the onlythree statins contained in the bank. These data show that it is possibleto use MSC cells derived from iPS cells for high throughput screenings.The latter cells have functional characteristics very similar to MSCcells derived from embryonic stem cells.

B) Conclusions

The differentiated MSC cells derived from human induced pluripotentcells have a sensitivity to statins of the same type as the MPC cellsand are cell tools suitable for high throughput screening techniques forproducts having toxicity for muscle tissue. With this type of cells, thecell test developed for high throughput screening is both robust andsensitive. It should be added that it is possible to conduct this testunder conditions of a synthetic and defined medium.

Example 5 High Throughput Screening and Metabolism of Mevalonate:Applications for Muscle Atrophy and for Cancer

A) Introduction

In this series of tests, the goal is to isolate molecules which maymodify the toxic effect of inhibitors of HMG CoA reductase. In this way,it will therefore be possible to sort out the molecules on thecapability of reducing or specifically enhancing the toxic effects ofinhibitors of HMG CoA reductase.

The molecules which reduce the toxic effect may be associated withinhibitors of HMG CoA reductase in order to reduce the deleteriouseffects of the latter on muscle tissue. On the other hand, thesemolecules may have more extensive applications in the protection ofmuscle atrophy. Indeed, it was shown that in the large majority ofmuscle atrophies, the atrigon protein is increased and this regardlessof the origin of muscle atrophy. Further, the treatment of the cells byinhibitors of HMG CoA reductase increases the synthesis of the atrogin.It is therefore legitimate to believe that the cells treated byinhibitors of HMG CoA reductase may form an in vitro cell model ofmuscle atrophy. Muscle atrophy is an extremely common pathology which isassociated with many pathologies (neuromuscular diseases, cancer orHIV). In the latter pathologies, atrophy is a poor prognosis factor.Preventing or reducing muscle atrophy is therefore a significanttherapeutic goal.

The molecules enhancing the toxic effect are of interest for more thanone reason. Knowing these molecules might give the possibility ofavoiding their therapeutic combinations with inhibitors of HMG CoAreductase in order to prevent potential toxic effects. On the otherhand, the inhibitors of HMG CoA reductase are used in combination withanti-cancer molecules for increasing their therapeutic potentials inanti-tumoral treatment. The definition of the molecules enhancing theeffects of inhibitors of HMG CoA reductase should give the possibilityof proposing a rational basis for defining therapeutic combinations fortreatments of tumoral diseases.

B) Principles for the Screening of the Bank of Small Molecules withDifferentiated MSC Cells Derived from Pluripotent Cells Treated withInhibitors of HMG COA Reductase

MSC cells derived from pluripotent cells are sown in the presence of aninhibitor of HMG CoA reductase and treated with the bank of smallmolecules. In this experiment, the inhibitor of HMG CoA reductase usedwas simvastatin at a concentration of 2 μM. After 72 hours ofcultivation, the number of cells is determined for each well by the CellTiter Glow technique. In order to demonstrate the feasibility of thetechnique, the Prestwick bank of small molecules was selected, whichcontains more than 1,200 compounds, for which the large majority ofactive ingredients are used in human clinical practice like for Example2. The working process is schematized in Annex 2.

C) Results of the Screening on Differentiated MSC Cells Derived fromHuman Pluripotent Cells Treated with Simvastatin.

The screening conditions were:

-   -   2,000 cells per well among 384 wells    -   D+1 treatment with compounds from the Prestwick bank    -   D+1 treatment with simvastatin    -   D+4 read out of the results with Cell Titer Glow.

With the screening, it is possible to identify two types of compounds.The first has a protective role and limits the toxicity of inhibitors ofHMG CoA reductase for MSC cells. The second exacerbate toxicity of thestatins.

The selections of the thresholds was carried out by calculating theaverage of the samples and the standard deviation. With thismethodology, a series of protective compounds and a series of compoundsexacerbating toxicity of simvastatin were determined.

For the first compounds, the following results were obtained which areshown in Table 14.

TABLE 17 NUMBER OF COMPOUNDS HAVING LARGER AVERAGES M + 1ET M + 2ET M +2.5ET M + 3ET M + 4ET M + 5ET M + 6ET Number 105 11 4 3 2 1 0 of hits M= Average of the samples ET = standard deviation Number of hits = numberof molecules having an average above the average + N standard deviations

11 compounds have a larger average than that with two standarddeviations and only 2 compounds have a larger average than that with 4standard deviations. The thereby selected compounds belong to differentdrug classes (antihypertensives, antihistaminics, andtidepressors oranticholinergics). By using this approach, it was therefore possible toisolate compounds which allow reduction in the toxic effect of statins.One of the first applications of the thereby isolated molecules will beto prevent the deleterious effects of statins and thereby extend theirclinical indications. On the other hand, these molecules may have aprotective role in muscle atrophy, a very common pathology for whichtherapeutic tools are still too reduced. Indeed, MSC cells treated withinhibitors of the synthesis of mevalonate represent a model in theculture dish for muscle atrophy observed in many primitive pathologies(muscle dystrophy) or secondary pathologies (sarcopenia, cancers, HIV,ageing). This type of screening will allow definition of drug strategiesfor reducing muscle atrophy.

For the second series of compounds which enhance the effect of statins,the following results were obtained which are shown in Table 15.

TABLE 18 Number of compounds having lower averages M − 1ET M − 2ET M −3ET M − 4ET M − 5ET M − 6ET Number of hits 650 203 59 42 32 27 M =Average of the samples ET = Standard deviation Number of hit = number ofmolecules having an average of less than the average + N standarddeviations

32 compounds have a lower average by 5 standard deviations. Among these32 compounds, unexpectedly three statins present in the bank are found.Indeed, the selected concentration of simvastatin is sub-optimum andunder these conditions, it is expected that the statins present in thebank will be detected. For finer analysis, it is possible to isolatecompounds for which toxicity is enhanced in the presence of simvastatin.Indeed among the 32 compounds, 5 compounds were not revealed in toxicitytests in the absence of sinvastatin. It is therefore possible to isolatecompounds which specifically increase the observed toxicity in thepresence of a statin. Among these molecules, anti-tumoral molecules andmolecules which have recognized muscular toxicity are found. With thistest, it is possible to predict the molecules which enhance the tissuetoxicity due to statins. With this approach, it is possible to preventtoxicities due to drug interactions in the presence of statins. Thissimple functional test to be set into place may therefore be practicedsystematically for all novel molecules before their passing intoclinical practice.

The screening strategy is the following. The first screening allowsisolation of the molecules which enhance toxicity in the presence ofcells treated with statins. Counter-screening is then performed on thethereby isolated molecules from the first screening in order to removethe molecules which have toxicity in the absence of statins. In thisway, the specificity of the toxicity in the presence of statins isensured. It is significant that, with this strategy among the 5 sortedout molecules, 3 are antitumoral molecules.

C) Conclusions

The developed cell test (bioassay) which uses as a cell source,differentiated MSC cells derived from pluripotent cells treated with aninhibitor of HMG Coa reductase, Simvastatin, allows sorting out of themolecules which protect from the toxic effect of simvastatin and of themolecules which exacerbate this effect.

The first families of molecules may have therapeutic applications forlimiting the toxic effects of inhibitors of HMG Coa reductase and forpreventing muscle atrophy, a major pathology.

The second families of molecules may have toxicological applications forpredicting undesirable drug interactions and therapeutic applications intumoral treatment for determining the molecule families which may becombined with inhibitors of HMG Coa reductase for obtaining cytotoxiceffects on tumoral cells and on <<tumoral stem>> cells.

The invention claimed is:
 1. A method for selecting pharmaceuticalcompounds that enhance inhibition of mevalonate synthesis and enhancecell toxicity in the presence of inhibitors of the synthesis ofmavalonate, the method comprising: providing mesodermal stem cells (MSCtype) that are derived from human pluripotent cells or from induced stemcells; contacting the MSC type cells with pharmaceutical compounds inpresence of an inhibitor of mevalonate synthesis; and selectingpharmaceutical compounds that enhance inhibition of mevalonate synthesisand enhance cell toxicity in the presence of inhibitors of the synthesisof mavalonate.
 2. A method for selecting pharmaceutical compoundsaccording to claim 1, wherein the cells of the MSC type obtained frominduced stem cells are cultivated in the presence of 1% of fetal calfserum.
 3. A method for selecting pharmaceutical compounds according toclaim 1, wherein the MSC type cells are contacted with an inhibitor ofHMG CoA reductase, simvastatin or all compounds of this drug class.
 4. Amethod for selecting pharmaceutical compounds according to claim 1,comprising a first screening that allows isolation of the pharmaceuticalcompounds which enhance cell toxicity in the presence of cells treatedwith statins and then a counter-screening is performed on the therebyisolated pharmaceutical compounds from the first screening in order toremove the pharmaceutical compounds which have toxicity in the absenceof statins.
 5. A method for selecting pharmaceutical compounds accordingto claim 1, wherein the human pluripotent cells or induced stem cellsare obtained in a culture medium comprising a) one or more growthfactors selected from FGFs, HGF, PDGFs, EGF, herugulins and VEGFs, andb) one or more antioxidants selected from ascorbic acid and itsderivatives, vitamin E and N-acetylcysteine.